Abstract -- Although mass vaccination programs have resulted in the eradication of a number of human infectious diseases, vaccine contamination has been a persistent concern. In particular, it is now known that the early polio vaccines were contaminated with at least one monkey virus, SV40. The transfer of monkey viruses to man via contaminated vaccines is particularly relevant to the acquired immunodeficiency syndrome (AIDS), since the causative agent of AIDS, human immunodeficiency virus (HIV), is thought to be derived from a simian precursor virus. Furthermore, human infection with this virus appears to be a relatively recent event. We hypothesize that the AIDS pandemic may have originated with a contaminated polio vaccine that was administered to inhabitants of Equatorial Africa from 1957 to 1959. The mechanism of evolution of HIV from this vaccine remains to be determined.

Date received 21 September 1992 Date accepted 5 January 1993

Introduction

The development of vaccines against infectious diseases has been a boon to mankind. For example, the global eradication of smallpox was announced by the World Health Assembly in May 1980. This long-dreaded disease was defeated with a vaccination program that extended throughout the poorest countries and reached the most inaccessible areas of the world (1). There are now vaccines that are effective in preventing such viral infections as rabies, yellow fever, poliovirus and hepatitis B. Eventually, vaccines will probably prevent malaria, some forms of heart disease and cancer. Even venereal diseases may someday be a target of vaccination programs.

Modern vaccine technology originated with the work of Pasteur in the late 19th century. Pasteur created his successful vaccine against rabies by weakening the virulence of the rabies virus, repeatedly passing virus-laden saliva from the mouths of rabid dogs through the brains of rabbits. The rabbit brains were then allowed to age in glass bottles. Graded doses of emulsions made from the infected rabbit brains were given in a series of injections to people bitten by rabid animals. The injections gave these individuals immunity to the virus before disease symptoms had a chance to appear (1).

Pasteur's technique was the first step in the giant strides of vaccine technology that would lead to health improvements for the future. However along with the benefits of vaccines would come some serious new risks. Some of these risks were tragically unforeseen. In 1902, 19 Punjabi villagers given an experimental plague vaccine died of a tetanus contaminant (2); and in 1906 an American scientist in the Philippines inoculated 24 prisoners with an experimental cholera vaccine that inadvertently had been contaminated with plague. 13 of the men died (3).

The Pasteur technique was used in 1936 by Dr Max Theiler of the Rockefeller Institute to create a vaccine against yellow fever. To produce the new vaccine, virus strains obtained from infected individuals were passed through the tissues of mice instead of rabbits. Fertilized chicken eggs were then seeded with these weakened yellow fever viruses. After a week of incubation, the chick-embryos were removed from the eggs and finely minced. Human blood serum was then added to stabilize the viruses. In 1938 more than one million Brazilians were inoculated with the vaccine before it was discovered that it had been contaminated with hepatitis B virus (1). Despite this disaster, human blood serum continued to be used as a stabilizer in yellow fever vaccines until 1942, when approximately 330 000 people came down with hepatitis B virus infection linked to vaccine lots given to approximately 50 000 US Army personnel. There were at least 84 deaths from the 1942 hepatitis outbreak (4).

However, the largest vaccine contamination in medical history occurred from 1954 through early 1963, when millions of people around the world received polio vaccines that had been contaminated with a monkey virus.

Killed and live polio vaccines

The road to the successful Salk and Sabin polio vaccines was a difficult one. The first attempt at a killed polio vaccine ended in complete failure. In 1935, a young researcher at the New York University School of Medicine isolated a poliovirus strain and injected it into monkeys. He then ground up the spinal cords of the infected monkeys and put the tissues into formaldehyde, which was supposed to kill the virus. The researcher then inoculated monkeys, as well as hundreds of children, with the 'killed' virus. When some of the vaccinated monkeys were challenged with live poliovirus, however, they promptly died of the disease. Investigation then revealed that at least 1 child had died and 3 others had become paralyzed after receiving the 'killed' vaccine (5).

It was thought for some time that, since laboratory signs of poliovirus infection were found in the nervous system, the virus could only be grown in nerve tissue. After the 1935 disaster, scientists were afraid to use monkey nervous tissue to make a killed vaccine. Then it was discovered that the poliovirus could grow in monkey kidneys. This finding allowed Dr Jonas Salk to begin producing polio vaccines with factory-like efficiency in 1952. Eventually, the laboratories making these vaccines would consume 200 000 monkeys a year (6).

As in 1935, formaldehyde was used by Salk to kill the polioviruses that he had isolated. Initial tests of his vaccine showed efficacy in preventing disease upon challenge of inoculated laboratory animals with live virus. The largest testing of a medical product in the history of man was then organized by the March of Dimes organization (5). But just before the mass public inoculations began in 1954. a worrying incident occurred. Monkeys at the National Institutes of Health (NIH) collapsed and died after receiving an injection of the Salk vaccine. Scientists, however, were relieved to discover upon postmortem examination that the monkeys had not died of polio but of some other disease frequently found in monkeys (5).

The Salk vaccine trials were interrupted again when it was discovered that vaccine lots produced by the Cutter Company had caused some monkeys and a total of 250 children and their contacts to develop complete or partial paralysis. 11 of the victims died. The Cutter product was hurriedly withdrawn, and the vaccine trial continued. It was later determined that bottles of tissue culture fluid containing the virus had been stored at Cutter before going through the formaldehyde-inactivation process. Bits of monkey kidney tissue debris had then settled to the bottom of the containers and covered virus particles, protecting them from the formaldehyde (5).

Like Salk with his killed vaccine, Dr Albert Sabin had to isolate viable virus from polio victims in order to develop his live oral polio vaccine. The virus strains had to be potent enough to cause immunity when ingested, but not so strong as to return to virulence after undergoing Pasteur's method of repeatedly infecting laboratory animals and harvesting the weakened viruses. Sabin grew his viruses in monkey kidney tissues, as Salk had done. But unlike Salk, he did not treat the viruses with formaldehyde (5).

Beginning in 1956, Sabin's live polio vaccine was tested in the Soviet Union and Eastern Europe by the administration of sweet syrup and sugar cubes to over 77 000 000 people. The live oral vaccine was then adopted in 1962 as the polio vaccine of choice for the United States and most of the world (5).

The discovery of Simian Virus 40 (SV40)

Euphoria at the triumph over the crippling disease of polio came to an abrupt end among some members of the scientific community when it was discovered in 1960 that both the Salk and Sabin vaccines had been contaminated. As a result, 10 to 30 million Americans and hundreds of millions of other people world-wide had been exposed to a simian virus called SV40 (7). This virus was found to produce a latent infection in monkey kidneys and had a cancer-causing potential, as indicated by its ability to produce tumors in laboratory animals. Tumors caused by SV40 in these animals were often sarcomas occurring at the site of inoculation, but were also found in kidneys and lungs. 3-week-old hamsters infected with SV40 produced a wide variety of tumors, most of which were lymphomas and bone cancers. SV40 was also discovered to transform human cells in vitro, and the transformed cells could then produce localized tumors when injected back into the human donors (7).

Initially, there was no definite evidence that SV40 was active in humans. Even as late as 1975, the journal Science wrote:

Who could have argued against the benefits of polio vaccine in the 1950s -- yet the vaccine received by millions of people in the United States and abroad is now known to have been contaminated with SV40, a monkey virus which causes tumors in hamsters, though not, as luck would seem to have it, in man. (8)

However, by then SV40 had been isolated from the brains of 2 patients with progressive multifocal leukoencephalopathy (PML) (9) and from an advanced melanoma.(10). Moreover, an Australian study demonstrated a correlation between polio immunization and the development of cancers in children over 1 year of age (11). In other reports, footprints of SV40 were found in adult and pediatric brain tumors (12), (13), and an increased occurrence of intracranial tumors was noted among persons who had received the contaminated vaccines (14). SV40 was also implicated in the development of bladder, oromaxillofacial, and parotid gland tumors (15), (16), (17). More recently, it has been discovered that endothelial cells transformed by SV40 cause Kaposi sarcoma-like tumors in immunodeficient mice (18), and that latent SV40 infection can be reactivated by simian immunodeficiency virus (SIV) to cause kidney cancers and PML in monkeys (19). Yet despite these findings, no major studies of the possible consequences of the massive population exposure to SV40 have been conducted to date.

However, in 1988, a study conducted between 1959 and 1965 in 58 807 pregnant women was reviewed (20). Data from this Collaborative Perinatal Project demonstrated that the risk of brain tumors among offspring of mothers who had received the Salk vaccine was 13 times the risk among offspring of mothers who had not. The stored serum samples of the mothers of offspring with cancers were tested for antibodies to SV40. Despite the association between the vaccine and the occurrence of brain tumors in vaccinee offspring, none of the mothers' sera were positive (20). The conclusion of the reviewers was intriguing: the cancers were probably caused by a still-unidentified infection originating in the polio vaccine. which (according to the reviewers) was known to have been contaminated with numerous simian viruses (21).

What happened when SV40 was discovered in the vaccines? The Director of the Division of Biologics Standards of the NIH issued a memorandum to manufacturers of the live oral polio vaccine on June 30, 1961, ordering them to exclude SV40-contaminated lots from all vaccines used in the United States (7). Since the Asian monkeys used in vaccine production were up to l00% infected with SV40, the manufacturers began to import large quantities of African green monkeys which did not naturally harbor the virus or show antibodies to it upon capture in the wild (22). Thus vaccine production switched from predominantly Asian monkeys to African green monkeys in 1961.

A new human disease emerges

Acquired immunodeficiency syndrome (AIDS) is a pandemic disease of high mortality afflicting all countries of the world. The majority of cases reported to date have been in North America, Western Europe, Equatorial African and Brazil. The pandemic is also rapidly spreading among other Third World countries such as Thailand and India, and has reached such high levels in parts of Africa that some countries are now threatened with negative population growth.

AIDS first came to the attention of medical researchers in early 1981 when the Centers for Disease Control in Atlanta, Georgia, reported some unusual infections and cancers occurring among homosexual men in New York, Los Angeles and San Francisco. Epidemiologists then began looking for more cases in other cities around the United States. What they discovered was that the first cases of the disease in the homosexual community had probably occurred in New York City in 1978 (23).

Also occurring in New York City's homosexual community that same year was the first large scale clinical trial of a new vaccine against hepatitis B virus. This vaccine was derived from the blood of healthy human carriers of the virus. The New York City Blood Center trials were placebo-controlled, double-blind randomized tests conducted in 1083 male homosexual volunteers (1). Had this medical experiment anything to do with the outbreak of AIDS in the homosexual community? Since the 1000 medical workers in other parts of the country were also given the experimental vaccine and had not come down with AIDS, the timing was thought to be only coincidental.

By the fall of 1981, AIDS had apparently spread beyond the homosexual communities. The Montefiore Hospital in the Bronx began treating cases in heterosexual intravenous drug users, and the New York and New Jersey state health departments reported that some prison inmates had the tell-tale opportunistic infections. The Jackson Memorial Hospital in Miami then reported that Haitians were coming down with the disease. Soon hemophiliacs and recipients of blood transfusions were also dying of it (23).

In October 1983, French physicians reported that a deadly disease almost identical to AIDS was raging in Equatorial Africa. It was readily apparent that a contagion was causing the deaths. A breakthrough in the mystery came when Drs F. Barre-Sinoussi and Luc Montagnier of the Pasteur Institute in Paris discovered an unusual new retrovirus in the blood of two victims. A year later. US researchers led by Dr Robert C. Gallo confirmed the French finding of the retrovirus implicated in AIDS. The new retrovirus was called human immunodeficiency virus (HIV) (23).

The notion that AIDS was a new disease in the Western hemisphere was supported by the fact that no hemophiliac in the United States had died from the disease before 1980, even though blood from Haiti was often used to produce the coagulant factor that the patients' lives depended on (23). That the disease was also a recent occurrence in the rest of the world was shown by the fact that the earliest detection of HIV in the tissues of a European was in a British sailor who died in Manchester, England in 1959 (24). The earliest known serum sample containing antibodies against HIV also dates from that same year (25). The serum was collected from an unknown patient visiting a clinic in Leopoldville, the Belgian Congo (now Kinshasa, Zaire). There is no laboratory evidence of HIV infecting humans before 1959 (26).

Simian AIDS

In February 1983, veterinary scientists made a startling discovery. Monkeys at the University of California Primate Research Center and at Harvard's New England Primate Center were suffering waves of illnesses strikingly similar to those seen in AIDS patients. These monkey epidemics actually had begun in 1969, but were not thought of as significant (monkeys often died in captivity) until the recognition of AIDS in 1981 (23).

A retrovirus which was 40% identical to HIV was soon isolated from an ailing macaque monkey. It was called simian immunodeficiency virus (SIV). The monkey had probably been infected with SIV while in captivity, since it was discovered that the natural hosts of SIV were African green monkeys, just as Asian macaque monkeys had been found in 1960 to be the natural hosts for SV40 (27).

The discovery of a virus related to HIV occurring naturally in the monkey species that was preferred for vaccine production caused the World Health Organization (WHO) to convene two 'informal' meetings of experts in 1985. At the time, the conclusions issued by WHO seemed reassuring: first, live polio vaccines prepared in African green monkey kidney cultures during the 1970s had been tested for retroviruses using reverse transcriptase assays and electron microscopy, and (given the nature of the tests) none had been found; second, WHO had tested vaccine seed stocks as well as 20 batches of vaccine for retroviruses, and again (given the tests used) none had been found. In addition, WHO had checked 250 vaccine recipients for HIV antibodies, and none were positive. 30 of these recipients were also tested for SIV antibodies, and all were negative. Finally, WHO said that long-term follow-up of vaccine recipients had shown no sign of adverse effects potentially associated with a retrovirus (28).

Apprehensions were revived, however, when it was discovered that some West Africans were infected with a virus that resembled SIV (29). The virus identified in their blood was called HIV-2. Like HIV-1, it was soon implicated in the development of AIDS. Researchers from the Japan Poliomyelitis Research Institute then undertook their own investigation of vaccine contamination. They found that approximately 26% of the African green monkeys used in vaccine production in Japan had antibodies against SIV. They killed 2 of these monkeys and looked for the virus, but couldn't isolate it in the monkey kidneys -- though they readily found it in blood, bone marrow, spleen, tonsils and lymph nodes. Vaccine stocks were then tested, and again (given the tests used) no SIV was found. In addition, no antibodies to the virus were detected in 190 vaccine recipients. However, the conclusion of the Japanese researchers was that more caution should be exercised. They recommended that monkeys infected with SIV should not be used in the preparation of vaccines (30). And sure enough, in May 1991, it was reported that researchers using more sensitive tests for SIV had found virus DNA in virtually all of the tissues and organ systems of infected monkeys, including the kidneys (31). Furthermore, a SIV not previously known to infect humans was recovered from the cancer cells of an AIDS patient (32) and SIV infection has now been discovered in laboratory workers, agricultural workers and urban dwellers (33), (34).

To some researchers. there appeared little doubt that human AIDS had its origins in the recent cross-species transfer of African monkey viruses to man (35), and to others that this transfer took place via contaminated vaccines (36), (37), (38), (39), (40). Since SIV was quite different from HIV-1, however, it was unclear exactly how this cross-species transfer could occur through vaccines. In 1990, 2 wild chimpanzees in Africa were discovered to be infected with a strain of SIV that was 75-84% identical to HIV-1 (41), leading some researchers to call it 'the missing link' to the origins of HIV-1 in man (42). It was thought that the chimpanzees may have been infected through contact with an unknown monkey species (23). This finding gave no comfort to those who disputed the vaccination theory, since chimpanzees had been used to attenuate and test viruses for potential use in vaccines and were often kept in captivity by vaccine laboratories (43), (44). Chimpanzees, therefore, could be a source of vaccine contamination and infection of other captive monkeys. It is now known that HIV can infect at least 1 species of macaque monkey (45), and HIV antibodies have been detected in captive African green monkeys (46).

Because of the size of the current epidemic in Africa and because of HIV-positive serum showing up there as early as 1959, it is now generally agreed that AIDS originated in Africa (23). But if contaminated polio vaccines were responsible for the introduction of HIV to man, why was its early occurrence so geographically localized and not more widely distributed? Certainly vaccination programs such as the Salk vaccine trial in the United States and the Sabin trials in the Soviet Union and Eastern Europe were affecting millions of people around the world. If the vaccines were linked to the origin of AIDS, why were Africans the first to come down with the disease? Perhaps the answer is that Africans were not immune to polio vaccine trials.

The Congo vaccine

In the 1950s there were other researchers besides Salk and Sabin who were racing to win the polio vaccine honors. One of them was an American who worked for one of the largest pharmaceutical manufacturers in the world. Before attempting to develop an oral polio vaccine, this researcher had done studies on yellow fever virus in Brazil (47). Like Salk and Sabin, he had to perform successful isolation of poliovirus strains, weaken the viruses by infecting humans and laboratory animals with them, test them for virulence, and then make the viruses proliferate in culture. His supervisor at the pharmaceutical company preferred using fertile chicken eggs instead of monkey kidney cultures (he was afraid of the unseen dangers), but kidney cultures were more effective in growing polioviruses, and so the researcher began to use them in conjunction with chick embryo cultures (48).

The problem that the researcher faced was in testing his live polio vaccines. His first attempt to inoculate humans came to light in March 1951, when he announced at a medical conference that he had given his vaccine in chocolate milk to a group of mentally defective children in an institution in New York State (49). When the Salk vaccine trials began, the researcher had to find a population in another country in order to test his product. He chose Belfast, Northern Ireland. But his vaccine trial in Belfast was stopped when his weakened viruses appeared to return to virulence. He then left the pharmaceutical company and went to head a research institute, announcing, however, that he would continue to develop a live polio vaccine (5).

In 1955, the researcher attended a rabies course organized by the World Health Organization in Kenya. There he met the director of a medical laboratory in Stanleyville, Belgian Congo. He proposed to the laboratory director a program of experiments administering a new polio vaccine to chimpanzees. The director agreed, and a colony of chimpanzees was created in Lindi Camp, Belgian Congo, for the American researcher's use. The animal keepers were inoculated with the new vaccine, supposedly 'to protect them' from the experiments with the chimps. The successful inoculation of the animal keepers was the excuse then used to propose a mass public inoculation program (30).

From 1957 to 1959, the American researcher's vaccine was given to hundreds of thousands of inhabitants of the Belgian Congo, including the area which now comprises Kinshasa and eastern Zaire as well as the countries of Rwanda and Burundi. Over 320 000 of the vaccine recipients were infants and children (49). In a preliminary report in the British Medical Journal of July 26, 1958, the American researcher and his colleagues printed a detailed map of the areas in which residents had been inoculated with the Congo vaccine (49). This map correlates with another map provided by authors of a report 30 years later identifying areas of high levels of HIV infection in Equatorial Africa (51). Another study in 1985 concluded that HIV infection among adult residents of this area had probably occurred in childhood (52).

In 1958, another polio vaccine researcher studied the particular strain of attenuated poliovirus used in the Congo vaccine. He discovered that the strain was contaminated with an 'unidentified non-poliomyelitis virus' (53). In response, the American researcher wrote that all vaccines made in monkey tissues were probably contaminated with unknown simian viruses (54), (55). In addition, because of an embargo in India that affected the monkey trade beginning in 1955, some of the kidneys used in producing the vaccine may have been obtained from African green monkeys or other simian species (6). Thus the origin of contaminating virus, as well as the exact nature of the manufacture of the vaccine, is uncertain (56), (57), (58).

Because of independence and resulting tribal chaos and civil war breaking out in the Belgian Congo in 1960, there was no long-term follow-up of this mass inoculation. The American researcher claimed that he had the permission of the World Health Organization to conduct the trial, but WHO later denied it (5). Since the Congo vaccine was never approved for human use, it was never used after 1960. (Sabin won the live vaccine honors.) When WHO tested seed stocks of poliovirus for HIV and SIV in 1985, had it also tested seed stocks of this researcher's viruses? And when WHO tested actual polio vaccines in 1985, had his 1957 Congo product been one of them?

It is now known that HIV can readily be transmitted through mucosal tissues (59). The 1957-1959 inoculations in the Congo were performed by squirting live polio vaccines from syringes into the mouths of the vaccinees (60). This procedure would have aerosolized some of the liquid and been a very efficient mode of HIV transmission. Assuming that the average time between infection with HIV and development of AIDS symptoms is 8-10 years (as is currently believed), the first outbreaks of disease occurring in Africa and related to this vaccine would have taken place in the period from 1965 to 1971. Is it only a coincidence that this is exactly the same period when scientists now believe that AIDS began occurring in Equatorial Africa (61)?

It is difficult to believe that the outbreak of HIV infection in Africa at the same time and location as this mass polio vaccine trial is a coincidence. But medical scientists also assumed that it was a coincidence when the first cases of AIDS in the homosexual community in New York followed the first hepatitis B vaccine trial in that community, just as NIH scientists believed in 1954 that some monkeys falling down and dying of a monkey disease after receiving the Salk-vaccine was a coincidence.

Conclusions

Whether the 1957-59 polio vaccine inoculations in the Belgian Congo were the cause of the cross-species transfer of HIV to man remains to be proven. What we do know is that the Congo vaccine was passaged in monkey tissue, that it was contaminated with at least one unidentified non-poliomyelitis virus, and that it was given to hundreds of thousands of people (including infants and children) in an area that is now endemic for HIV disease. But instead of acknowledging the possible role of medical science in the origin of the AIDS pandemic, some researchers have been throwing stones at the first victims. Among theories expounded to explain the African genesis of AIDS are: Africans eating monkeys; Africans keeping monkeys as pets; and Africans engaging in rituals in which monkey blood is used as a magic potion (51), (62), (63). But Africans have engaged in these practices for thousand of years, while AIDS is an entirely new disease. Is it only a coincidence that HIV infection manifested itself at the same time as the introduction of vaccines that are now known to have been contaminated with simian viruses? Whatever the case, as one scientist has written: 'The story of AIDS teaches us that animal tissues should not be injected into humans, because the risk of introducing a new virus is too great' (63).

Acknowledgements

The authors wish to acknowledge Peggy Tahir and Kathy Kimber for research assistance, and Ann Giudici Fettner and Tom Curtis for helpful discussion. We also thank Steven Koontz for expert technical assistance.

After we independently developed our hypothesis and original manuscript in September, 1991, we discovered that Louis Pascal had already proposed a similar hypothesis about the Congo vaccine (64). We wish to acknowledge Mr Pascal's pioneering work (65).

References

1. Chase A. Magic Shots. New York: William Morrow and Company Inc., 1982.

Polio Vaccines and the Origin of AIDS: Clarification

Correspondence

Dear Sir

Our article on polio vaccines and the origin of AIDS (Medical Hypotheses 1994; 42: 347-354) has provoked extensive discussion of issues related to the origin and spread of human immunodeficiency virus (HIV). We wish to address two of these issues.

Our article failed to mention the fact that the Congo poliovirus vaccine trials began as early as February, 1957 (1). The Manchester sailor who presumably died of AIDS in 1959 purportedly did not return to England until the 'first half of 1957' (2). Therefore, the possibility exists that the sailor could have been exposed to the HIV-like contaminant that we hypothesize may have been transmitted in the Congo vaccine. The sailor's rapid disease progression and death from P. carinii pneumonia may have been due to the fact that he was unwittingly treated with massive doses of corticosteroids by his physicians (3).

Further research has also identified the fact that sexually active homosexuals in the United States did not initially receive the same hepatitis B virus (HBV) vaccine that was given to US medical workers. Homosexual men were recruited not only for participation in the HBV vaccine trials but also for serum donation of the HBV subtype antigen adw that was given to the homosexual volunteers (4). In the trials involving medical workers, however, an entirely different HBV antigen subtype obtained from dialysis patients (ayw) was used (5). Formaldehyde was employed for inactivation of HBV in the vaccine given to homosexual men (6). The same inactivation method was used by Salk for his 'killed' poliovirus recipients (7). Thus, the control and sterilization methods used in the HBV vaccine trials are open to question.

Finally, we wish to point out two typographical errors among the references in our article. On page 351, second column, second paragraph, the reference cited as number 30 should be number 50. Likewise, the preliminary report in the British Medical Journal of July 26, 1958, cited in the next paragraph should also be reference number 50.